Molecular Devices ImageXpress Micro User manual

5014166 D
September 2015
ImageXpress®Micro
High Content Imaging System
Options
User Guide
ImageXpressOptionsUserGuide.book Page 1 Wednesday, September 9, 2015 3:54 PM

This document is provided to customers who have purchased Molecular Devices
equipment, software, reagents, and consumables to use in the operation of such
Molecular Devices equipment, software, reagents, and consumables. This document
is copyright protected and any reproduction of this document, in whole or any part,
is strictly prohibited, except as Molecular Devices may authorize in writing.
Software that may be described in this document is furnished under a non-
transferrable license. It is against the law to copy, modify, or distribute the software
on any medium, except as specifically allowed in the license agreement.
Furthermore, the license agreement may prohibit the software from being
disassembled, reverse engineered, or decompiled for any purpose.
Portions of this document may make reference to other manufacturers and/or their
products, which may contain parts whose names are registered as trademarks and/or
function as trademarks of their respective owners. Any such usage is intended only to
designate those manufacturers’ products as supplied by Molecular Devices for
incorporation into its equipment and does not imply any right and/or license to use
or permit others to use such manufacturers’ and/or their product names as
trademarks.
Each product is shipped with documentation stating specifications and other
technical information. Molecular Devices products are warranted to meet the stated
specifications. Molecular Devices makes no other warranties or representations
express or implied, including but not limited to, the fitness of this product for any
particular purpose and assumes no responsibility or contingent liability, including
indirect or consequential damages, for any use to which the purchaser may put the
equipment described herein, or for any adverse circumstances arising therefrom. The
sole obligation of Molecular Devices and the customer's sole remedy are limited to
repair or replacement of the product in the event that the product fails to perform as
warranted.
For research use only. Not for use in diagnostic procedures.
Product manufactured by Molecular Devices, LLC.
1311 Orleans Drive, Sunnyvale, California, United States of America 94089.
Molecular Devices, LLC is ISO 9001 registered.
© 2015 Molecular Devices, LLC.
All rights reserved.
The trademarks mentioned herein are the property of Molecular Devices, LLC or their respective owners. These
trademarks may not be used in any type of promotion or advertising without the prior written permission of
Molecular Devices, LLC.
Patents: http://www.moleculardevices.com/productpatents
ImageXpressOptionsUserGuide.book Page 2 Wednesday, September 9, 2015 3:54 PM

5014166 D 3
Contents
Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Who this User Guide is For. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Conventions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Obtaining Support. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Chapter 1: Environment Control Option
Operations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Environment Control Hardware. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Items Included in the Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Items to Be Provided by the Customer. . . . . . . . . . . . . . . . . . . . . . 11
Setting Up Environmental Control. . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Setting the Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Setting Up the Water Reservoir . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Setting Up the Carbon Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Loading the Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Environment Control Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Environmental Parameters in Image Information . . . . . . . . . . . . . 19
Performing Timelapse Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . 20
Reviewing Timelapse Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Chapter 2: Fluidics Option Operations . . . . . . . . . . . . . . . . . . . . 27
Fluidics Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Items to Be Provided by the Customer. . . . . . . . . . . . . . . . . . . . . . 29
Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Setting Up Fluidics Hardware. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Configuring Fluidics Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Manual Fluidics Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Performing Fluidics Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Preventing Evaporation from Compound Plates . . . . . . . . . . . . . . . 43
Evaluated Seals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
ImageXpressOptionsUserGuide.book Page 3 Wednesday, September 9, 2015 3:54 PM

ImageXpress Micro High Content Imaging System Options User Guide
4 5014166 D
Chapter 3: Environment Control and Fluidics
Options Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Carbon Dioxide Tubing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Cleaning the Water Reservoir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Waste Disposal Box and Plate Sealing Ring . . . . . . . . . . . . . . . . . . . . 47
Chapter 4: Transmitted Light Option Operations . . . . . . . . . . . 49
Transmitted Light Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Transmitted Light Software Configuration . . . . . . . . . . . . . . . . . . . . 54
Transmitted Light Phase Ring Alignment . . . . . . . . . . . . . . . . . . . . . . 56
Transmitted Light Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
ImageXpressOptionsUserGuide.book Page 4 Wednesday, September 9, 2015 3:54 PM

5014166 D 5
Preface
Who this User Guide is For
This user guide is written for those who want to use an ImageXpress®
Micro High Content Imaging System with any of the following optional
components:
•ImageXpress Environment Control option
•ImageXpress Fluidics option
•ImageXpress Transmitted Light option
The ImageXpress Environment Control option, ImageXpress Fluidics
option, and ImageXpress Transmitted Light option described in this guide
are available for only the ImageXpress Micro High Content Imaging
System.
Conventions
In this user guide, the following typographical conventions are used.
CAUTION! Indicates an operation that can cause damage to the
instrument, device, or data, if the precautions are not followed.
.
WARNING! A warning indicates an operation that can cause personal
injury if precautions are not followed.
Note: Provides essential information for the completion of a
procedure.
Tip: Provides useful information that helps apply the techniques and
procedures in the text to your specific needs, and can also provide
shortcuts. The information in a tip is not essential to the completion of a
procedure.
ImageXpressOptionsUserGuide.book Page 5 Wednesday, September 9, 2015 3:54 PM

ImageXpress Micro High Content Imaging System Options User Guide
6 5014166 D
MetaXpress Software
Use the MetaXpress®Software with the ImageXpress Micro System to
select a standard image analysis routine or to develop a custom protocol
to fit your specific acquisition and analysis needs. The MetaXpress
Software workflow is divided into two major parts: acquisition and
analysis.
•The acquisition workflow involves configuring settings, acquiring
images, and storing plate data in a database.
•The analysis workflow consists of processing, enhancing, and
analyzing acquired plate data. See the MetaXpress High Content
Image Acquisition & Analysis Software Analysis Guide included
on the MetaXpress Software installation USB flash drive.
Simplified Menu Structure
An optional simplified menu structure can be installed to reduce the
number of top-level menus in the MetaXpress Software. All the features
of the software are available in this reorganized menu structure.
The procedures in this guide describe both the default menu structure
and the simplified menu structure.
You can use the Menu Map in the Help menu to help you find the
locations of features in the simplified menu structure. The Menu Map is
available only after the simplified menu installation.
1. Click Help > Menu Map.
2. In the Menu Map dialog, select to view the Default to
customized menu map.
3. Click the menu path where the software feature you want is
found in the default menu structure.
The simplified menu path appears to the right of the desired
feature in the menu.
4. Click the menu path in the software window to access the
desired feature.
For example, if you want to make a duplicate of an image, then use the
following procedure:
1. Click Help > Menu Map.
2. In the Menu Map dialog, select to view the Default to
customized menu map.
3. Click Edit > Duplicate.
The simplified menu path -> Edit: Image: Duplicate Image/Plane
appears to the right of the Image option in the submenu.
ImageXpressOptionsUserGuide.book Page 6 Wednesday, September 9, 2015 3:54 PM

Preface
5014166 D 7
4. In the software window, click Edit > Image > Duplicate
Image/Plane.
Obtaining Support
Molecular Devices is a leading worldwide manufacturer and distributor
of analytical instrumentation, software, and reagents. We are committed
to the quality of our products and to fully supporting our customers with
the highest possible level of technical service.
Our support web site, http://www.moleculardevices.com/support, has a
link to the Knowledge Base with technical notes, software upgrades,
safety data sheets, and other resources. If you do not find the answers
you are seeking, follow the links to the Technical Support Service Request
Form to send an email message to a pool of technical support
representatives.
You can contact your local representative or contact Molecular Devices
Technical Support by telephone at 800-635-5577 (U.S. only) or
[phone]. In Europe call [phone].
Part of effective communication with Molecular Devices is determining
the channels of support for the ImageXpress Micro System, including the
MetaXpress Software. Molecular Devices provides a wide range of
support:
•Documentation: Check the guides that are included on the
installation media and the help that is available within the
MetaXpress Software. Help for an active dialog can be accessed
by pressing F1 on your keyboard.
•Online knowledge base: The knowledge base has links to
technical notes, software upgrades, newsletters, user guides,
and other resources. Visit the Molecular Devices Support web
page at http://www.moleculardevices.com/support and follow
the links to the knowledge base.
•MetaMorph Software forum: This forum has information on
journal scripts and custom modules, and has links to videos and
webinars that can help you troubleshoot problems and be more
productive using the software. Visit the forum at
metamorph.moleculardevices.com/forum.
ImageXpressOptionsUserGuide.book Page 7 Wednesday, September 9, 2015 3:54 PM

ImageXpress Micro High Content Imaging System Options User Guide
8 5014166 D
•Technical Support:
Phone: Contact Technical Support at (800)-635-5577 (U.S. only)
or [phone]. In Europe call [phone].
Online: Visit http://www.moleculardevices.com/support and
follow the links in the knowledge base to the Technical Support
Request Form to send an email to a group of experienced
Technical Support representatives.
Please have the system ID number, system serial number,
software version number, and the name of the system owner
available when you call.
To find your system ID number, in the MetaXpress Software,
click Help > About MetaXpress. The About dialog displays
your system ID number.
The system serial number is located on your instrument.
•Additional support resources include:
Nikon web-based microscopy course:
http://www.microscopyu.com
The Molecular Probes Handbook:
http://www.lifetechnologies.com/us/en/home/references/
molecular-probes-the-handbook.html
This resource offers advice on fluorescent probes and can
help you determine if there are better stains available for
your analysis.
The following sites offer filter information:
- http://www.chroma.com
- http://www.semrock.com
- http://www.omegafilters.com
ImageXpressOptionsUserGuide.book Page 8 Wednesday, September 9, 2015 3:54 PM

5014166 D 9
1
Environment Control Option Operations
The ImageXpress®Environment Control option is designed to maintain an
environment for living cells to enable multi-day live-cell timelapse
imaging. Temperature, carbon dioxide, and humidity can all be
maintained within the sample plate so that cells can be kept alive for
many days, growing at a rate comparable to that expected in a standard
cell culture incubator. In addition to offering kinetic and timelapse
imaging capabilities, the environmental enclosure can accommodate a
single-channel fluidics robot for delivering compounds during
experimentation.
The ImageXpress Environment Control option can be installed together
with either the Fluidics option or the Transmitted Light option.
See also:
•Fluidics Option Operations on page 31
•Transmitted Light Option Operations on page 55
This chapter contains the following sections:
•Environment Control Hardware on page 10
•Setting Up Environmental Control on page 14
•Environment Control Software on page 21
•Doing Timelapse Experiments on page 23
ImageXpressOptionsUserGuide.book Page 9 Wednesday, September 9, 2015 3:54 PM

ImageXpress Micro High Content Imaging System Options User Guide
10 5014166 D
Environment Control Hardware
The ImageXpress Environment Control option consists of a sealing ring
on top of the sample plate and a top door above the plate that together
forms a small, sealed volume. Humidified carbon dioxide is sourced into
this small volume to form the required environment above the plate.
Temperature is controlled within the upper half of the instrument.
The ImageXpress Environment Control option consists of the following
hardware subsystems:
•Temperature control within the upper half of the base
instrument. Warm air is provided from the Environmental
Control Option Controller or the Systems Power & Options
Controller through an air hose. Feedback from temperature
sensors installed near the plate maintains the temperature. The
temperature can be controlled within a range of 30°C to 40°C,
when the ambient room temperature is 22°C or lower.
•Carbon dioxide is provided from a customer-supplied tank of
pre-mixed 5% CO2and 95% air. The tank regulator must be set
between 15 PSI and 20 PSI. The Environmental Control Option
Controller or the Systems Power & Options Controller controls
the flow to the space above the plate, maintained by the live-cell
sealing ring. If a plate is ejected and loaded, the system conducts
a purge cycle automatically.
•Humidity is passively provided by bubbling the carbon dioxide
through a water reservoir, minimizing evaporation from the
sample plate over the duration of a timelapse experiment.
Items Included in the Installation
The following hardware components are included in an ImageXpress
Environment Control option installation:
•ImageXpress Micro Environmental Control Option Controller or
the ImageXpress Micro Systems Power & Options Controller.
See Figure 1-1 on page 11 and Figure 1-2 on page 11.
•Warm air hose and carbon dioxide tubing. See Figure 1-3 on
page 12 and Figure 1-4 on page 12.
•Temperature sensors. To ensure accurate readings, the sensors
are located near the sample plate.
•Water reservoir. See Figure 1-5 on page 13.
•Live-cell sealing ring. Compatible with 96-well and 384-well
standard height plates. The standard height for these plates is
14.35 mm ± 0.25 mm (0.5650 inches ± 0.0098 inches). See
Figure 1-6 on page 13.
ImageXpressOptionsUserGuide.book Page 10 Wednesday, September 9, 2015 3:54 PM

Environment Control Option Operations
5014166 D 11
Figure 1-1: Environmental Control Option Controller used for the
ImageXpress Micro Standard and XLS Systems
Figure 1-2: Systems Power & Options Controller used for the
ImageXpress Micro Confocal System
Note: The Compound Plate Temperature setting is available only when
the Fluidics option is also installed.
ImageXpressOptionsUserGuide.book Page 11 Wednesday, September 9, 2015 3:54 PM

ImageXpress Micro High Content Imaging System Options User Guide
12 5014166 D
Figure 1-3: Environment Control option warm air hose and carbon
dioxide tubing on the back of the Environmental Control Option
Controller
Figure 1-4: Environment Control option warm air hose and carbon
dioxide tubing on the back of the ImageXpress Micro Standard System
ImageXpressOptionsUserGuide.book Page 12 Wednesday, September 9, 2015 3:54 PM

Environment Control Option Operations
5014166 D 13
Figure 1-5: Environment Control option water reservoir
Figure 1-6: Environment Control option live-cell sealing ring
ImageXpressOptionsUserGuide.book Page 13 Wednesday, September 9, 2015 3:54 PM

ImageXpress Micro High Content Imaging System Options User Guide
14 5014166 D
Items to Be Provided by the Customer
The customer must provide the following items for the Environment
Control option installation:
•A tank of pre-mixed 5% CO2and 95% air.
•The regulator, fittings, and tubing required to deliver 15 to 20 PSI
carbon dioxide from the tank to the Environmental Control
Option Controller or the Systems Power & Options Controller.
•Microwell plates with standard ANSI height. Most 96-well and
384-well plates are standard height. The standard height for
these plates is: 14.35 mm ± 0.25 mm (0.5650 inches ± 0.0098
inches).
•Deionized water to maintain humidity.
Setting Up Environmental Control
An FSE (Field Service Engineer) installs the ImageXpress Environment
Control option. After installation, environmental control can be set up for
experiments.
Setting the Temperature
The temperature controller is calibrated before the instrument ships
from the factory. Use the external temperature controller to set the
temperature that you want the environmental enclosure to maintain.
ImageXpressOptionsUserGuide.book Page 14 Wednesday, September 9, 2015 3:54 PM

Environment Control Option Operations
5014166 D 15
To set the temperature
1. On the front of the Environmental Control Option Controller or
the Systems Power & Options Controller, view the current
sample plate temperature.
Figure 1-7: Sample Plate Temperature control interface
2. To view the temperature set point, press .
3. To increase the temperature set point, press and .
4. To decrease the temperature set point, press and .
After you release , the current temperature in the chamber is
displayed.
Setting Up the Water Reservoir
Air supplied to the environmental enclosure flows through the water
reservoir to make sure the air has a high degree of humidity.
Tools required for setup:
Syringe
Deionized Water (preferably sterilized)
Note: For the ImageXpress Micro Confocal System, you can set the
temperature with the Instrument power switch in the On position. To
regulate the temperature in the chamber, the Sample Heater power
switch must be also in the On position.
ImageXpressOptionsUserGuide.book Page 15 Wednesday, September 9, 2015 3:54 PM

ImageXpress Micro High Content Imaging System Options User Guide
16 5014166 D
To set up the water reservoir
1. In the MetaXpress software, click Screening > Plate acquisition
and Control > Eject Plate to open the top door of the instrument.
2. Remove the insert that surrounds the top door.
3. Disconnect the CO2/air tubing from the luer lock on the rear of
the instrument.
4. Insert a syringe filled with deionized water into the luer lock.
5. View the water reservoir through the open top panel.
Figure 1-8: Environment Control option water reservoir
6. Dispense the deionized water from the syringe into the water
reservoir until the water reaches the fill line.
7. Remove the syringe from the luer lock.
8. Reconnect the CO2/air tubing to the luer lock.
9. Replace the insert that surrounds the top door.
10. In the MetaXpress software, click Screening > Plate acquisition
and Control > Load Plate to close the top door of the instrument.
ImageXpressOptionsUserGuide.book Page 16 Wednesday, September 9, 2015 3:54 PM

Environment Control Option Operations
5014166 D 17
Setting Up the Carbon Dioxide
A pre-mixed CO2 tank with a regulator must be set up and connected to
the carbon dioxide controller. The carbon dioxide controller then controls
the flow rate of carbon dioxide delivered to the water reservoir within
the environmental enclosure.
Before you begin, make sure the water reservoir is set up. See Setting Up
the Water Reservoir on page 15.
To set up the carbon dioxide
1. Connect the tubing from the pre-mixed CO2 tank to the
Environmental Control Option Controller or the Systems Power &
Options Controller.
2. Verify that the tubing from the Environmental Control Option
Controller or the Systems Power & Options Controller to the
instrument is connected.
3. Connect the regulator to the CO2tank.
4. Turn on the CO2regulator to approximately 15 PSI to 20 PSI.
5. From the top of the ImageXpress Micro System, lift the insert
surrounding the top door and remove it. Keep the top door of
the instrument closed.
6. Verify that there is a steady flow of bubbles in the water
reservoir.
7. Replace the insert surrounding the top door.
8. Check the environmental control settings to verify that the CO2
pressure is OK. See Environment Control Software on page 21.
After setting up the carbon dioxide
Load an unlidded plate with the live-cell sealing ring on top. If there are
concerns about contamination, a breathable seal can be used over the
top of the plate. See Loading the Sample Plate on page 18.
Note: Before you do imaging experiments, wait for the system and
plate to reach equilibrium. Allow at least two hours for the system and
30 minutes for the plate. Because focus settings and offsets change with
temperature, you might need to optimize them after the system and
plate have reached equilibrium.
ImageXpressOptionsUserGuide.book Page 17 Wednesday, September 9, 2015 3:54 PM

ImageXpress Micro High Content Imaging System Options User Guide
18 5014166 D
Loading the Sample Plate
You must use the live-cell sealing ring to maintain the CO2flow to the
sample plate. The sealing ring helps to contain the inputted air directly
over the cells and maintains the proper CO2and temperature levels.
To load the sample plate
1. In the MetaXpress software, click Screening > Plate acquisition
and Control > Eject Plate to open the top door of the instrument.
2. Insert the sample plate into the stage and then remove the lid
from the plate. If there are concerns about contamination, a
breathable seal can be used over the top of the plate.
WARNING! BIOHAZARD! Wear gloves when handling sample plates.
Note: To ensure that it is at the proper temperature, before you load
the sample plate, make sure that the live cell sealing ring is in the system
or in the incubator.
ImageXpressOptionsUserGuide.book Page 18 Wednesday, September 9, 2015 3:54 PM

Environment Control Option Operations
5014166 D 19
3. Place the live-cell sealing ring directly on top of the plate, making
sure that it fits securely onto the plate.
Figure 1-9: Environment Control option live-cell sealing ring
4. In the MetaXpress software, click Screening > Plate acquisition
and Control > Load Plate to close the top door of the instrument.
Note: Temperature fluctuations in the plate and its
surroundings cause the plate and its cells to shift in X,Y, and Z
direction. To minimize these temperature fluctuations, take the
following precautions.
ImageXpressOptionsUserGuide.book Page 19 Wednesday, September 9, 2015 3:54 PM

ImageXpress Micro High Content Imaging System Options User Guide
20 5014166 D
Before you run an experiment:
•Allow the instrument to warm up for at least two hours.
•Make sure the plate sealing ring is at 37°C before use, either by
keeping it inside the instrument or inside an incubator. Incorrect
sealing-ring temperature can cause temperature fluctuation.
•Wait 30 minutes to 1 hour for the plate to reach equilibrium. You
can use a journal to set this waiting period.
The current temperature in the chamber is displayed on the
front of the controller.
•If there is interstitial space between wells, pipette deionized
water or media into these areas. This helps to increase the
thermal mass of the plate and reduces overall evaporation.
1.
Note: Due to the changes in Z height over time, you might need
to adjust the plate focus parameters in the MetaXpress software
occasionally. To do so, open the MetaXpress software, and then
click Screening > Plate Acquisition Setup > Plate tab > Edit Plate
Bottom Settings. Increase the adjacent well max variation to
accommodate the added Z-height variation introduced by the
fluctuation of temperature.
When setting laser autofocus options, make sure that the
instrument and plate temperatures are at equilibrium. Room-
temperature laser autofocus settings are not ideal when the
system is used at higher temperatures.
ImageXpressOptionsUserGuide.book Page 20 Wednesday, September 9, 2015 3:54 PM